A functionally essential domain of RFX5 mediates activation of major histocompatibility complex class II promoters by promoting cooperative binding between RFX and NF-Y

Major histocompatibility complex class II (MHC-II) molecules occupy a pivot al position in the adaptive immune system, and correct regulation of their expression is therefore of critical importance for the control of the immun e response. Several regulatory factors essential for the transcription of M HC-II genes have been identified by elucidation of the molecular defects re sponsible for MHC-II deficiency, a hereditary immunodeficiency disease char acterized by regulatory defects abrogating MHC-II expression. Three of thes e factors, RFX5, RFXAP, and RFXANK, combine to form the RFX complex, a regu latory protein that binds to the X box DNA sequence present in all MHC-II p romoters. In this study we have undertaken a dissection of the structure an d function of RFX5, the largest subunit of the RFX complex. The results def ine two distinct domains serving two different essential functions. A highl y conserved N-terminal region of RFX5 is required for its association with RFXANK and RFXAP, for assembly of the RFX complex in vivo and in vitro, and for binding of this complex to its X box target site in the MHC-II promote r. This N-terminal region is, however, not sufficient for activation of MHC -II expression. This requires an additional domain within the C-terminal re gion of RFX5. This C-terminal domain mediates cooperative binding between t he RFX complex and NF-Y, a transcription factor binding to the Y box sequen ce of MHC-II promoters. This provides direct evidence that RFX5-mediated co operative binding between RFX and NF-Y plays an essential role in the trans criptional activation of MHC-II genes.